Imaging repair processes of small epithelial lesions in the mouse trachea after laser-induced injury

Small lesions in the airway epithelium occur frequently and offer an entry for pathogens. Therefore injuries have to be repaired to prevent prolonged disruption of epithelial integrity and to maintain a normal airway function. The mechanism of the repair process of small lesions in the airways is largely unknown.

To better understand this repair process of small lesions in the airways we used two-photon microscopy and an ex-vivo model of the mouse trachea. The explanted trachea was cut longitudinally and then imaged with the epithelium facing up. Epithelial lesions were induced by focussing Ti-sapphire femtosecond laser pulses to single epithelial cells for 1 – 8 seconds. Staining with propidium iodide (PI) allowed identification of damaged cells. Phalloidin was used to stain actin filaments after the experiment.

Damaging of cells in a specific area of the epithelium was possible. Depending on the irradiation time an area of 1 – 12 epithelial cells was damaged.

Hyperfluorescence around the beam focus and loss of autofluorescence in adjacent cells was observed. Within the lesion nuclei were stained with PI. Small lesions of 1 – 3 cells were closed within 2 – 3h. Lesions of 4 – 6 cells needed 4 – 5h or did not close within the observation time of up to 6h. Epithelial cells around the lesion changed their shape by stretching. Especially the cells immediately adjacent to the wound margin protracted notably to close the lesion. During this process damaged cells were expelled apically into the lumen. Staining with phalloidin after two-photon microscopy showed a transepithelial actin ring formed in the cells around the wound which participate in closing the lesion.

Healing of small lesions in the airways depends on an active coordinated movement of adjacent epithelial cells. Further studies will focus on the molecular mechanisms of repair that might be impaired in airway diseases.